Project summary Lipid droplets are intracellular fat storage organelles that play critical roles for lipid and energy homeostasis in both health and disease. Lipid droplets also have a second major function as regulators of protein homeostasis: droplets store specific proteins from other cellular compartments, participate in protein refolding and turnover, and serve as assembly platform for viruses. While there has been great progress in unraveling how droplets control lipid metabolism, studies of mechanisms and physiological relevance of their protein- handling roles remain limited. Yet a better understanding of these processes will not only illuminate many fundamental cellular processes, but may also inform treatment of the myriad of human diseases characterized by over- or understorage of fat. The goal of this project is to mechanistically dissect two proposed general functions of protein sequestration by lipid droplets: long-term storage to safely preserve excess levels of otherwise harmful or unstable proteins and short-term buffering to equalize the availability of proteins despite fluctuations in biosynthesis or demand. This application focuses on an extensively validated example of protein sequestration: histone storage on lipid droplets in early Drosophila embryos. Three key features make this a promising model: First, a crucial component of the sequestration machinery has been defined molecularly, the histone anchor Jabba. Second, buffering activity undergoes a dramatic developmental transition, providing an opportunity to probe the regulation and biological significance of buffering. Third, it is possible to combine classical and molecular genetics, biochemistry, proteomics, and live imaging to study sequestration in an intact, developing organism. Previous work has identified histone- and lipid-droplet binding activities in Jabba. This information will now be employed to determine how these activities promote histone storage, in particular whether Jabba protects histones from degradation or promotes their efficient delivery. Characterization of Jabba variants and photoactivation experiments will determine whether Jabba acts solely as static histone anchor or can accompany histones in the cytoplasm. Using proteomics, post-translational modifications and binding partners of Jabba (and possibly other components) will be determined before and after the transition, with the goal of manipulating the transition and testing the consequences when buffering is turned on or off at inappropriate times. Finally, Jabba is known to promote dispersal of lipid droplets throughout the embryo. The mechanistic basis for this role will be determined, using structure-function approaches and analysis of a putative Jabba binding partner. These investigations will make it possible to test if droplet dispersal enhances buffering or serves other functions, such as modulating lipid metabolism. If successful, these studies will provide paradigms for how lipid droplets can employ regulated protein sequestration to control processes in other compartments of the cell.